Characterization of the Composition and Biological Activity of the Venom from Vespa bicolor Fabricius, a Wasp from South China.

We analyzed, for the first time, the major components and biological properties of the venom of Vespa bicolor, a wasp from South China. Using HPLC and SDS-PAGE, combined with LC-MS/MS, MALDI-TOF-MS, and NMR data to analyze V. bicolor venom (VBV), we found that VBV contains three proteins (hyaluronidase A, phospholipase A1 (two isoforms), and antigen 5 protein) with allergenic activity, two unreported proteins (proteins 5 and 6), and two active substances with large quantities (mastoparan-like peptide 12a (Vb-MLP 12a), and 5-hydroxytryptamine (5-HT)). In addition, the antimicrobial activity of VBV was determined, and results showed that it had a significant effect against anaerobic bacteria. The minimum inhibitory concentration and minimum bactericidal concentration for Propionibacterium acnes were 12.5 µg/mL. Unsurprisingly, VBV had strong antioxidant activity because of the abundance of 5-HT. Contrary to other Vespa venom, VBV showed significant anti-inflammatory activity, even at low concentrations (1 µg/mL), and we found that Vb-MLP 12a showed pro-inflammatory activity by promoting the proliferation of RAW 264.7 cells. Cytotoxicity studies showed that VBV had similar antiproliferative effects against all tested tumor cell lines (HepG2, Hela, MCF-7, A549, and SASJ-1), with HepG2 being the most susceptible. Overall, this study on VBV has high clinical importance and promotes the development of Vespa bicolor resources.

GH and GHR gene cloning, expression and their associations with growth-related traits of the barbel chub (Squaliobarbus curriculus).

Growth hormone (ScGH) and growth hormone receptor (ScGHR) genes from the barbel chub (Squaliobarbus curriculus), in addition to their cDNAs, were cloned. The associations between their mRNA expression levels and growth-related traits were analysed, and the differences in the levels of expression of growth regulation-related genes between the largest and smallest individuals were compared. The full-length 1182-bp cDNA of ScGH contained a 633-bp open reading frame (ORF), and the length of the gene had 2492 bp. The full-length 2825-bp cDNA of ScGHRa contained a 1818-bp ORF, and the gene had 6970 bp. The full-length 2822-bp cDNA of ScGHRb contained a 1737-bp ORF, and the gene had 8149 bp. Quantitative real-time PCR revealed that ScGH was only expressed in the pituitary. ScGHRa was expressed predominantly in muscle, and the expression level of ScGHRb was the highest in the liver. The ScGHRa mRNA levels in the muscle were significantly negatively correlated with the caudal peduncle length. However, no correlation between growth-related traits and ScGH and ScGHRb expression levels were found. Pituitary ScGH, liver GHRb and liver insulin-like growth factor I (igf-1) expression levels were significantly higher in the largest individuals than those in the smallest S. curriculus individuals. Contrarily, the largest individuals had significantly lower expression levels of muscle igf-1 and liver myog than the smallest individuals. Overall, our results provide novel molecular information for growth-regulation study of S. curriculus.

Characterization and immune function of the interferon-ß promoter stimulator-1 in the barbel chub, Squaliobarbus curriculus.

To elucidate the immunity-protecting role of the interferon-ß promoter stimulator-1 (ScIPS-1) in barbel chub Squaliobarbus curriculus, the full-length cDNA of ScIPS-1 was cloned and expression levels in response to stimulation were investigated. In addition, the function of ScIPS-1 and its domains were analyzed. The full-length cDNA of ScIPS-1 is 2524 bp and encodes 601 aa. The N-terminal caspase activation and recruitment domain, central proline-rich domain, C-terminal transmembrane domain, C2HC-zinc finger, and Cwf21 domains were identified. The mRNA level of ScIPS-1 was the highest in the kidney, whereas the highest protein level was observed in the liver. The ScIPS-1 expressions were significantly up-regulated after lipopolysaccharide and poly I:C treatment. The ScIPS-1 protein level was up-regulated at 12 h in the head kidney and was up-regulated at 12 h and then down-regulated from 12 to 48 h in the liver after grass carp reovirus (GCRV) infection. The CiIFN and CiMx transcription levels were significantly enhanced in pEGFP-C1-IPS-1 and pcDNA3.1-ΔCwf21 overexpressing cells after GCRV infection. The results indicate that ScIPS-1 may function in the immune response against pathogens and provide a basis for achieving resistance to diseases in fish breeding.

Molecular cloning and expression analysis of coagulation factor VIII and plasminogen involved in immune response to GCRV, and immunity activity comparison of grass carp Ctenopharyngodon idella with different viral resistance.

The grass carp reovirus (GCRV) has been shown to cause lethal infections in the grass carp Ctenopharyngodon idella (C. idella). In order to investigate the immune response to GCRV infection, the full-length cDNA sequences of coagulation factor VIII (CiFVIII) and plasminogen (CiPLG) from C. idella were cloned and their involvement in the immune response was studied. The immunity factor levels in C. idella with different GCRV resistances were also analyzed. The full-length 2478 bp cDNA of CiFVIII contained an open reading frame of 1965 bp and encoded a putative polypeptide of 654 amino acid residues. The full-length 2907 bp cDNA of CiPLG contained an open reading frame of 2133 bp and encoded a putative polypeptide of 710 amino acid residues. CiFVIII was closely clustered with that of Clupea harengus. CiPLG was first clustered with those of Cyprinus carpio and Danio rerio. CiFVIII transcripts were most abundant in the liver and least in the skin. The highest expression level of CiPLG was observed in liver and the lowest in muscle. Expression levels of CiFVIII in gill, head kidney and spleen, and expression levels of CiPLG in gill, intestine and liver all reached the maximum at 72 h post GCRV infection. In spleen, expression levels of CiFVIII and CiPLG were significantly positively correlated. The activities of T-AOC, LSZ and IgM in R♂ were significantly higher than those in O♂. Likewise, T-AOC and LSZ activities in F1 were significantly higher than f1 individuals (P < 0.01). These results indicated that CiFVIII and CiPLG may play important roles in the immune response to GCRV infection. In addition, antioxidant ability and serum immune factor activity may confer a different viral resistance to C. idella.

Molecular characterization and expression of TLR7 and TLR8 in barbel chub (Squaliobarbus curriculus): Responses to stimulation of grass carp reovirus and lipopolysaccharide.

The barbel chub (Squaliobarbus curriculus) is a kind of small size commercial fish species that is widely spread in Asia and has shown significant resistance to disease. In this study, the full-length cDNA sequences of Toll-like receptor (TLR) 7 and 8 from S. curriculus, designated as ScTLR7 and ScTLR8, were cloned, and their differences in the structure and the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation were investigated. The full-length 3715 base pair (bp) cDNA of ScTLR7 contained a complete open reading frame of 3162 bp and encoded a putative polypeptide of 1053 amino acid residues. The full-length 4624 base pair (bp) cDNA of ScTLR8 contained a complete open reading frame of 3072 bp and encoded a putative polypeptide of 1023 amino acid residues. ScTLR7 and ScTLR8 consisted of N-terminal signal peptide, leucine-rich repeats (LRRs), and Toll/IL-1 Receptors domain. LRR motifs of ScTLR7 and ScTLR8 bend into horseshoe-like solenoid structure, while the number of LRRs between the two genes is different. Phylogenetic analysis showed that both the ScTLR7 and ScTLR8 were closely clustered with Ctenopharyngodon idellus and Megalobrama amblycephala. Quantitative real-time polymerase chain reaction analysis showed that the expression levels of ScTLR7 in S. curriculus were most abundant in the brain followed by the spleen and heart, and the lowest in the intestine. The highest expression level of ScTLR8 was observed in spleen and the lowest in the liver. After LPS stimulation, the relative expression levels of both ScTLR7 and ScTLR8 exhibited an overall trend of up-regulation. The expression levels of type I-IFN showed an overall trend of down-regulation at time points of 12, 24, 72 and 168 h compared to that of 6 h after LPS stimulation. Compared to 6 h post GCRV infection, the transcription level of ScTLR7 was up-regulated from 12 to 168 h, and transcription levels of ScTLR8, MyD88, and type I-IFN were firstly up-regulated from 12 to 72 h, and then down-regulated from 72 to 168 h. Correlation analysis showed that expression level of ScTLR7 in the spleen was significantly positively correlated with that of MyD88 (Pearson correlation coefficient: 0.909, P: 0.033), and a significantly positive correlation was also observed between expression levels of MyD88 and type I IFN (Pearson correlation coefficient: 0.962, P: 0.009), after GCRV stimulation. These results indicate that ScTLR7 and ScTLR8 may play important roles in the responses to the grass carp (GCRV) infection and lipopolysaccharide stimulation and trigger different downstream immune signal pathways.

Atrial electrical remodeling in a canine model of sinus node dysfunction.

AIMS: To study atrial tachycardia-induced electrical remodeling in a canine model of sinus node dysfunction (SND). MATERIALS AND METHODS: A canine model of SND was established by contacting a cotton patch with 20% formaldehyde on the sinus node. Atrial effective refractory period (ERP), ERP dispersion, and inducibility of atrial fibrillation (AF) were recorded at multiple sites in the atrium, before and after SND induction as well as after rapid atrial pacing. The recovery of atrial ERP in the left and right atrium (LA and RA) after cessation of atrial pacing was also recorded. RESULTS: Compared with baseline, the atrial ERPs were shortened after SND (P<0.05). After rapid atrial stimulation, the atrial ERPs were further decreased significantly (P<0.05), and the dispersion of atrial ERPs measured at different pacing cycle lengths (PCLs) showed significant variation. Seven sites were used to induce AF in each dog (56 sites in 8 dogs). The average duration and inducibility of AF after SND was increased compared with baseline (16.5±4.7 vs 2.3±1.2 s and 12/56 vs 4/56 sites, P<0.05). After rapid atrial stimulation, the average duration and inducibility of AF were further increased (16.5±4.7 vs 33.6±16.1 s and 12/56 vs 25/56 sites, P<0.05). The recovery of atrial ERP in LA was significantly delayed compared to the RA. CONCLUSION: SND induces atrial electrical remodeling which is further aggravated by atrial tachycardia. Therefore, SND creates an electrophysiological substrate that facilitates AF initiation and perpetuation.

The continuous accumulation of Na+ in detached leaf sections is associated with over-expression of NTHK1 and salt tolerance in poplar plants.

Detached leaf sections (2×2cm2) from transgenic poplar line 18-1 and its wild type (WT) (Populus× euramericana 'Neva') were used to test their salt tolerance and gene expression under controlled environment conditions. The sections from line 18-1 displayed better tolerance to NaCl stress, indicated by high chlorophyll retention and K+ content but low relative electrolyte leakage (REL). Transient overexpression of NTHK1 (Nicotiana tabacum histidine kinase 1) and V-H+-PPase was found in the detached young leaves from line 18-1 after they had been stressed for a few minutes. The activities of vacuolar-type H+-ATPase and H+-PPase in line 18-1 were boosted initially and then decreased to normal level as in unstressed leaves. After sections were stressed for 10 days, the maximal Na+ concentration in line 18-1 was much higher than that in the WT. The higher capacity for Na+ accumulation in line 18-1 may be due to stable Na+ sequestration into the vacuoles. Osmotic stress imposed little effect on REL and chlorophyll content of the sections. The capacity of detached leaf sections in NaCl solution to tolerate stress and to accumulate Na+ may be useful for identifying genotypes with good salt tolerance in poplar and other plants.

[Standardization of technical methods for apple fluorescence canopy spectral detection].

Aiming at spectral detection of apple fluorescence canopy, the present paper carried out spectral detection tests under different weather conditions, different detection times, and different detection heights and angles to apple canopy in the two years of 2008 and 2009, so as to analyze impacts of these factors on apple canopy spectral characteristics and explore standardized spectral detection methods for apple fluorescence canopy. The results indicated the regularity in spectral reflectance of apple fluorescence canopy to a certain degree under different conditions, especially in the 760-1 350 nm near-infrared bands. The authors found that canopy spectral reflectance declined along with the decrease in sunshine and it is appropriate to detect canopy spectrum in sunny days with few clouds. In addition, spectral reflectance tended to be stable when the wind scale was below grade 2. The discrepancy of canopy spectra is small during the time period from 10:00 to 15:00 of a day compared to that of other times. For maintaining stable spectral curves, the height of detector to apple canopy needed to be adjusted to cover the whole canopy within the field of view according to detection angle of the detector. The vertical or approximately vertical detection was the best for canopy spectral reflectance acquisition. The standardization of technical methods of spectral detection for apple fluorescence canopy was proposed accordingly, which provided theoretical references for spectral detection and information extraction of apple tree canopy.